Project Summary Fetal-derived placenta cells are known to enter the maternal circulation during pregnancy and may persist in maternal tissue for decades as microchimeras. We have reported that fetal cells selectively home to injured maternal myocardium and undergo differentiation into diverse cardiac lineages. Using enhanced green fluorescent protein (eGFP)-tagged fetuses, we demonstrated engraftment and cardiac differentiation of mulitpotent fetal cells in injury zones of maternal hearts. In vitro, fetal cells isolated from maternal hearts recapitulate these differentiation pathways, forming vascular tubes and spontaneously beating cardiomyocytes in a fusion-independent manner. A significant proportion (~40%) of fetal cells in maternal hearts express Caudal-related homeobox2 (Cdx2), previously associated with trophoblast stem cells. Utilizing cre-lox technology for lineage-tracing, we have now shown that Cdx2 cells can be isolated from end-gestation placenta and can form beating cardiomyocytes and vascular cells in vitro. Furthermore, they exhibit a transcriptomic signature that suggests an ability to evade host immune surveillance. Proteomic studies of these cells compared to ES cells reveal distinct growth, survival and homing advantages, but with retention of the `stemness' properties of ES cells. Thus the transcriptomic and proteomic analysis reveal desirable qualities that can aid in the development of an allogeneic cell therapy approach. In further support of this objective, we have demonstrated that Cdx2 cells home robustly and specifically to infarcted hearts upon injection into the tail vein, with differentiation in vivo to cardiomyocytes and blood vessels. MRI demonstrates significant and sustained enhancement of contractility (manuscript in revision at PNAS). We have also shown that CDX2 cells can be isolated from human term placentas. Our final goal is to translate these studies for clinical use, and we propose three aims utilizing the most cutting-edge technologies in science in order to achieve this. In aim 1, we will uncover unique cell surface markers of human CDX2 cells via trancriptome/proteome profiling and explore their homing mechanisms. In aim 2, we seek to understand immunologic properties of these cells in order to aid in the development of allogeneic human cell therapy. We will also ascertain their propensity to form teratomas. In aim 3, we will confirm that CDX2 cells also give rise to functional cardiomyocytes and vascular cells in vitro and in vivo after myocardial infarction (MI) is induced in wild-type and NOD/SCID mice. We will also test whether these cells enhance myocardial function after MI, as this will aid us in designing a therapeutic strategy.